發(fā)布時(shí)間：2018-06-04 21:37

  本文選題：多形性膠質(zhì)母細(xì)胞瘤 + 多藥耐藥相關(guān)蛋白3　； 參考：《大連醫(yī)科大學(xué)》2012年博士論文

【摘要】：多形性膠質(zhì)母細(xì)胞瘤（GBM）是成人最常見、惡性度最高的原發(fā)腦腫瘤，WHO將其歸類為Ⅳ級星形細(xì)胞瘤。盡管手術(shù)技術(shù)不斷進(jìn)步，放療結(jié)合新一代化療藥物的應(yīng)用，GBM患者的預(yù)后仍然極差，中位生存期僅為15個(gè)月左右，迫切需要新的治療方法。近年提出針對腫瘤抗原的“抗體導(dǎo)向”治療新理念，以抗體為載體，將藥物導(dǎo)向腫瘤病灶，提高藥物療效，減少藥物毒副作用，代表腫瘤免疫治療的新方向。 多藥耐藥相關(guān)蛋白3（MRP3）在人類GBM實(shí)體瘤和細(xì)胞系中均呈高水平表達(dá)，且表達(dá)于腫瘤細(xì)胞膜表面。相反，MRP3在正常腦組織中不表達(dá)。因此，MRP3可作為抗體導(dǎo)向治療GBM的理想分子靶點(diǎn)。2010年，Kuan等利用噬菌體展示技術(shù)，分離得到抗MRP3的人源重組單鏈抗體（scFv），能夠與人類MRP3胞外區(qū)N端的氨基酸殘基特異性結(jié)合，是抗體導(dǎo)向治療GBM的理想抗體分子。 腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體（TRAIL），又稱凋亡素-2配體（APO-2L），屬TNF超家族成員，通過與死亡受體TRAIL-R1（DR4）和TRAIL-R2（DR5）結(jié)合，激活外源性凋亡途徑，誘導(dǎo)caspase依賴的細(xì)胞凋亡。TRAIL具有顯著的凋亡腫瘤細(xì)胞凋亡活性而對正常細(xì)胞沒有毒性，被視為最具有臨床應(yīng)用前景的抗癌藥物之一。天然TRAIL為膜結(jié)合型，其胞外區(qū)含有蛋白酶作用位點(diǎn)，可以從細(xì)胞膜上被剪切下來，或通過mRNA剪接作用，形成可溶性TRAIL（sTRAIL），仍然保留天然TRAIL的選擇性凋亡誘導(dǎo)活性。 本研究利用基因重組技術(shù)，首次將抗MRP3的單鏈抗體與僅對腫瘤細(xì)胞有很強(qiáng)殺傷作用的sTRAIL通過柔性氨基酸連接臂(Gly4-Ser)3相連，構(gòu)建抗MRP3的單鏈抗體與sTRAIL的融合蛋白，命名為antiMRP3(scFv)-sTRAIL，以期通過antiMRP3(scFv)與MRP3的特異結(jié)合，加強(qiáng)sTRAIL在GBM細(xì)胞膜表面的富集，達(dá)到靶向誘導(dǎo)GBM凋亡的目的。本論文將從以下三部分闡述antiMRP3(scFv)-sTRAIL融合蛋白對GBM的靶向凋亡誘導(dǎo)作用及其機(jī)制。 第一部分antiMRP3(scFv)-sTRAIL融合蛋白的誘導(dǎo)表達(dá)、純化及其生物學(xué)活性鑒定 利用pMAL原核表達(dá)系統(tǒng)誘導(dǎo)表達(dá)與純化antiMRP3(scFv)-sTRAIL融合蛋白，并鑒定其生物學(xué)活性。通過重組PCR技術(shù)分別擴(kuò)增antiMRP3(scFv)和sTRAIL基因片段，插入原核表達(dá)質(zhì)粒pMAL-c2，構(gòu)建重組表達(dá)質(zhì)粒pMAL-antiMRP3(scFv)-sTRAIL，經(jīng)菌落PCR、酶切鑒定及DNA測序證明構(gòu)建的重組質(zhì)粒與預(yù)期完全一致。將重組質(zhì)粒轉(zhuǎn)化感受態(tài)E.coli BL21，IPTG誘導(dǎo)表達(dá)，得到帶有麥芽糖結(jié)合蛋白（MBP）標(biāo)簽肽的antiMRP3(scFv)-sTRAIL融合蛋白，經(jīng)Amylose Resin親和層析柱純化，SDS-PAGE鑒定純化的融合蛋白。結(jié)果顯示，在92kD處觀察到純化的融合蛋白條帶。融合蛋白在E.coli BL21中的表達(dá)率約30%，純化的融合蛋白純度達(dá)95%。 以MRP3陽性U251多形性膠質(zhì)母細(xì)胞瘤細(xì)胞為研究對象，用MTT法分別測定不同濃度antiMRP3(scFv)-sTRAIL融合蛋白和MBP蛋白作用24h后對U251細(xì)胞的增殖抑制率，結(jié)果表明antiMRP3(scFv)-sTRAIL融合蛋白可顯著抑制U251細(xì)胞的增殖并呈濃度依賴性，而MBP蛋白無明顯作用。應(yīng)用IC50劑量（62.5nmol/L）的antiMRP3(scFv)-sTRAIL融合蛋白作用U251細(xì)胞24h后，倒置顯微鏡下觀察U251細(xì)胞形態(tài)的改變。antiMRP3(scFv)-sTRAIL融合蛋白處理組U251細(xì)胞在形態(tài)學(xué)上呈現(xiàn)明顯細(xì)胞凋亡改變，而MBP蛋白組和未處理組細(xì)胞未發(fā)生異常形態(tài)改變。 上述結(jié)果表明成功構(gòu)建了pMAL-antiMRP3(scFv)-sTRAIL重組質(zhì)粒，并實(shí)現(xiàn)了原核表達(dá)與純化antiMRP3(scFv)-sTRAIL融合蛋白。初步的生物學(xué)功能鑒定表明該融合蛋白具有顯著誘導(dǎo)U251多形性膠質(zhì)母細(xì)胞瘤細(xì)胞凋亡的活性，為進(jìn)一步研究其對U251細(xì)胞的靶向凋亡誘導(dǎo)作用和機(jī)制奠定了基礎(chǔ)。 第二部分antiMRP3(scFv)-sTRAIL融合蛋白對U251多形性膠質(zhì)母細(xì)胞瘤的靶向凋亡誘導(dǎo)作用 通過熒光激活細(xì)胞分選技術(shù)（FACS）分析antiMRP3(scFv)-sTRAIL融合蛋白與MRP3陽性U251細(xì)胞和MRP3陰性Jurkat細(xì)胞的抗原特異性結(jié)合。結(jié)果顯示antiMRP3(scFv)-sTRAIL融合蛋白可特異性結(jié)合至MRP3陽性U251細(xì)胞膜表面，其結(jié)合可被親代antiMRP3(scFv)競爭性阻斷，而antiMRP3(scFv)-sTRAIL融合蛋白與MRP3陰性Jurkat細(xì)胞未見明顯細(xì)胞膜表面結(jié)合，表明antiMRP3(scFv)-sTRAIL融合蛋白與U251細(xì)胞的結(jié)合為MRP3抗原特異性。 觀察應(yīng)用親代antiMRP3(scFv)競爭性阻斷antiMRP3(scFv)-sTRAIL與MRP3的靶向結(jié)合，或TRAIL活性中和抗體mAb2E5中和sTRAIL活性，對antiMRP3(scFv)-sTRAIL融合蛋白凋亡誘導(dǎo)活性的影響。用MTT法檢測應(yīng)用不同濃度antiMRP3(scFv)-sTRAIL作用于U251細(xì)胞24h后的細(xì)胞存活率，結(jié)果顯示antiMRP3(scFv)-sTRAIL呈現(xiàn)較強(qiáng)殺傷U251細(xì)胞作用，在24h內(nèi)隨融合蛋白濃度增加，U251細(xì)胞存活率逐漸降低。應(yīng)用antiMRP3(scFv)或mAb2E5預(yù)孵育后，antiMRP3(scFv)-sTRAIL對U251細(xì)胞的殺傷作用被顯著抑制，各濃度梯度下均無明顯的殺傷活性，MBP蛋白對U251細(xì)胞的存活率沒有影響。用流式細(xì)胞儀檢測IC50劑量（62.5nmol/L）的antiMRP3(scFv)-sTRAIL作用8h、12h、24h后U251細(xì)胞的凋亡情況，與親代antiMRP3(scFv)和未處理組對比得出，antiMRP3(scFv)-sTRAIL作用8h時(shí)U251細(xì)胞即發(fā)生明顯凋亡，24h內(nèi)細(xì)胞凋亡率隨誘導(dǎo)時(shí)間延長而上升。應(yīng)用antiMRP3(scFv)或mAb2E5預(yù)孵育，antiMRP3(scFv)-sTRAIL對U251細(xì)胞的凋亡誘導(dǎo)作用被完全阻斷，結(jié)果與MTT檢測相符。 上述結(jié)果表明，作為抗體導(dǎo)向的免疫毒素，antiMRP3(scFv)-sTRAIL融合蛋白僅與MRP3陽性U251細(xì)胞靶向結(jié)合并誘導(dǎo)其凋亡，其結(jié)合為MRP3抗原特異性。antiMRP3(scFv)-sTRAIL融合蛋白的凋亡誘導(dǎo)活性可被親代antiMRP3(scFv)競爭性抑制或被TRAIL活性中和抗體阻斷，由此證明antiMRP3(scFv)-sTRAIL通過antiMRP3(scFv)單鏈抗體導(dǎo)向部分特異性結(jié)合MRP3抗原，實(shí)現(xiàn)靶向誘導(dǎo)GBM細(xì)胞凋亡，其凋亡誘導(dǎo)活性是由sTRAIL部分介導(dǎo)的，而非靶向結(jié)合的antiMRP3(scFv)-sTRAIL不具備明顯凋亡誘導(dǎo)活性。 第三部分antiMRP3(scFv)-sTRAIL融合蛋白對TRAIL-R1和TRAIL-R2的激活作用 TRAIL-R1（DR4）和TRAIL-R2（DR5）是介導(dǎo)外源性細(xì)胞凋亡途徑的兩種死亡受體，，其中DR5在多種腫瘤中的表達(dá)水平高于DR4，因而在誘導(dǎo)腫瘤凋亡中處于主導(dǎo)地位。研究表明，天然膜結(jié)合型TRAIL可激活TRAIL-R1和TRAIL-R2兩種死亡受體，而可溶性sTRAIL只保留了對TRAIL-R1的激活作用。為分析antiMRP3(scFv)-sTRAIL對U251細(xì)胞兩種死亡受體的激活情況，用real-time PCR法首先分析U251細(xì)胞中TRAIL-R1和TRAIL-R2的mRNA表達(dá)水平，結(jié)果表明TRAIL-R1和TRAIL-R2在U251細(xì)胞中均呈陽性表達(dá)，其中TRAIL-R2的表達(dá)水平高于TRAIL-R1。用抗-TRAIL-R1和抗-TRAIL-R2阻斷抗體孵育U251細(xì)胞1h，分別單獨(dú)或同時(shí)阻斷TRAIL-R1和TRAIL-R2，然后應(yīng)用IC50劑量的antiMRP3(scFv)-sTRAIL處理U251細(xì)胞24h，流式細(xì)胞儀檢測細(xì)胞凋亡率，分析antiMRP3(scFv)-sTRAIL對兩種死亡受體的激活作用。結(jié)果顯示，阻斷TRAIL-R1凋亡率下降28%，阻斷TRAIL-R2凋亡率下降35%，同時(shí)阻斷TRAIL-R1和TRAIL-R2凋亡率下降46%。最后，用Western blot方法驗(yàn)證了antiMRP3(scFv)-sTRAIL融合蛋白激活外源性凋亡途徑，誘導(dǎo)caspase依賴的細(xì)胞凋亡。 上述結(jié)果表明，antiMRP3(scFv)-sTRAIL融合蛋白與靶抗原特異性結(jié)合將sTRAIL由可溶性形式轉(zhuǎn)變?yōu)樘烊籘RAIL的膜結(jié)合型形式，不僅能夠激活TRAIL-R1，而且恢復(fù)了其對TRAIL-R2的激活能力，增強(qiáng)sTRAIL的凋亡誘導(dǎo)活性，從而獲得增強(qiáng)的抗腫瘤作用。antiMRP3(scFv)-sTRAIL融合蛋白通過結(jié)合TRAIL-R1和/或TRAIL-R2兩種死亡受體，激活外源性細(xì)胞凋亡途徑，誘導(dǎo)caspase依賴的細(xì)胞凋亡。
[Abstract]:Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumor in adults. WHO is classified as grade IV astrocytoma. Although the surgical technique is progressing, radiotherapy combined with the application of a new generation of chemotherapeutic drugs, the prognosis of GBM patients is still very poor, the median survival time is only about 15 months, and a new treatment is urgently needed. In recent years, a new concept of "antibody oriented" therapy for tumor antigen is proposed. Using antibody as the carrier, it will guide the tumor to improve the efficacy of the drug, reduce the side effects of the drug, and represent the new direction of the tumor immunotherapy.
Multidrug resistance related protein 3 (MRP3) expressed high level in human GBM solid tumor and cell line, and expressed on the surface of tumor cell membrane. On the contrary, MRP3 was not expressed in normal brain tissue. Therefore, MRP3 can be used as an ideal molecular target for antibody directed treatment of GBM for.2010 years. Kuan and other uses phage display technology to isolate the human source of anti MRP3. Recombinant single chain antibody (scFv) can bind specifically to the amino acid residues at the N end of the extracellular domain of human MRP3, and is an ideal antibody molecule for antibody directed therapy of GBM.
The tumor necrosis factor related apoptosis inducing ligand (TRAIL), also known as apoptin -2 ligand (APO-2L), is a member of the TNF superfamily. By binding to the death receptor TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the apoptosis pathway is activated, and the apoptosis.TRAIL of the caspase dependent apoptosis.TRAIL has significant apoptotic cell apoptosis activity, but not to normal cells. Toxicity is considered as one of the most promising anticancer drugs. Natural TRAIL is a membrane binding type, and its extracellular domain contains protease action sites, which can be cut off from the cell membrane or through mRNA splicing to form soluble TRAIL (sTRAIL), and still retain the selective apoptosis inducing activity of Titian TRAIL.
In this study, we used gene recombination technology to combine the anti MRP3 single chain antibody with the sTRAIL only with the tumor cells which had a strong killing effect through the flexible amino acid connection arm (Gly4-Ser) 3, to construct a fusion protein of the single chain antibody against MRP3 and sTRAIL, named antiMRP3 (scFv) -sTRAIL, in order to combine antiMRP3 (scFv) with MRP3. To enhance the concentration of sTRAIL on the surface of GBM cell membrane to achieve the goal of targeting GBM apoptosis, this paper will explain the target apoptosis inducing effect and mechanism of antiMRP3 (scFv) -sTRAIL fusion protein on GBM from the following three parts.
Part one: induction, purification and biological activity identification of antiMRP3 (scFv) -sTRAIL fusion protein
AntiMRP3 (scFv) -sTRAIL fusion protein was expressed and purified by pMAL prokaryotic expression system, and its biological activity was identified. AntiMRP3 (scFv) and sTRAIL gene fragments were amplified by recombinant PCR technology, and the plasmid pMAL-c2 was inserted into the prokaryotic expression plasmid, and the recombinant expression plasmid pMAL-antiMRP3 (scFv) -sTRAIL was constructed. The recombinant plasmid was fully consistent with the expectation. The recombinant plasmid was transformed into the receptive E.coli BL21, IPTG was induced, and the antiMRP3 (scFv) -sTRAIL fusion protein with maltose binding protein (MBP) tagged peptide was purified by Amylose Resin affinity chromatography column, and the purified fusion protein was identified by SDS-PAGE. The results showed that the fusion protein was observed in 92kD. The purified fusion protein band was expressed. The expression rate of fusion protein in E.coli BL21 was about 30%, and the purity of purified fusion protein was 95%.
Using MRP3 positive U251 polymorphous glioblastoma cells as the research object, the proliferation inhibition rate of antiMRP3 (scFv) -sTRAIL fusion protein and MBP protein at different concentrations was measured by MTT method. The results showed that antiMRP3 (scFv) -sTRAIL fusion protein could significantly inhibit the proliferation and concentration dependence of the cells. The antiMRP3 (scFv) -sTRAIL fusion protein of IC50 dose (62.5nmol/L) acted on U251 cell 24h, and the morphology of U251 cells was observed under the inverted microscope. The apoptosis of U251 cells in the.AntiMRP3 (scFv) -sTRAIL fusion protein treated group was obviously changed in morphology, but the cells and the untreated groups did not occur. Abnormal morphologic changes.
The results showed that the recombinant plasmid of pMAL-antiMRP3 (scFv) -sTRAIL was successfully constructed and the prokaryotic expression and purification of antiMRP3 (scFv) -sTRAIL fusion protein was realized. The preliminary biological function identification showed that the fusion protein could induce the apoptosis of U251 multiform glioblastoma cells and further study its U251 cells. It lays the foundation for the inducing effect and mechanism of apoptosis.
The second part of antiMRP3 (scFv) -sTRAIL fusion protein induces apoptosis in U251 glioblastoma multiforme.
The antigen specific binding of antiMRP3 (scFv) -sTRAIL fusion protein with MRP3 positive U251 cells and MRP3 negative Jurkat cells was analyzed by fluorescence activated cell sorting (FACS). The results showed that the antiMRP3 (scFv) -sTRAIL fusion protein could be combined specifically to the surface of the MRP3 positive cell membrane. The combination of antiMRP3 (scFv) -sTRAIL fusion protein and MRP3 negative Jurkat cells showed no apparent adhesion to the cell membrane surface, indicating that the binding of antiMRP3 (scFv) -sTRAIL fusion protein with U251 cells was specific for MRP3 antigen.
The effects of antiMRP3 (scFv) on the targeting of antiMRP3 (scFv) -sTRAIL and MRP3, or the activity of TRAIL activity and the neutralization of the antibody mAb2E5 neutralization sTRAIL, on the apoptosis induced activity of antiMRP3 (scFv) -sTRAIL fusion protein were observed. The results showed that antiMRP3 (scFv) -sTRAIL showed a strong killing effect on U251 cells, and the survival rate of U251 cells decreased with the increase of fusion protein concentration in 24h. The killing effect of antiMRP3 (scFv) -sTRAIL on the cells was significantly inhibited after the incubation of antiMRP3 (scFv) or mAb2E5, and there was no obvious killing activity under the concentration gradient. P protein had no effect on the survival rate of U251 cells. The apoptosis of 8h, 12h and 24h U251 cells after antiMRP3 (scFv) -sTRAIL in IC50 dose (scFv) -sTRAIL was detected by flow cytometry. The induction time was prolonged and increased. The apoptosis induction of antiMRP3 (scFv) -sTRAIL on U251 cells was completely blocked by antiMRP3 (scFv) or mAb2E5 preincubation, and the results were consistent with the MTT detection.
These results suggest that as an antibody directed immuno toxin, antiMRP3 (scFv) -sTRAIL fusion protein only combines with MRP3 positive U251 cells and induces apoptosis. The apoptosis inducible activity of the MRP3 antigen specific.AntiMRP3 (scFv) -sTRAIL fusion protein can be suppressed competitively by antiMRP3 (scFv) or be neutralized by TRAIL activity and antibody. Blocking, it is proved that antiMRP3 (scFv) -sTRAIL can induce apoptosis of GBM cells by targeting specific binding of MRP3 antigen by antiMRP3 (scFv) single chain antibody, and its apoptosis induction activity is mediated by sTRAIL part, but the non targeting antiMRP3 (scFv) -sTRAIL does not possess obvious apoptosis induced activity.
The third part is the activation effect of antiMRP3 (scFv) -sTRAIL fusion protein on TRAIL-R1 and TRAIL-R2.
TRAIL-R1 (DR4) and TRAIL-R2 (DR5) are two kinds of death receptors that mediate exogenous apoptotic pathways, in which the expression level of DR5 is higher than that of DR4, so it is dominant in inducing tumor apoptosis. The study shows that the natural membrane binding TRAIL can activate the two death receptors of TRAIL-R1 and TRAIL-R2, and the soluble sTRAIL only remains. The activation of TRAIL-R1 was activated. In order to analyze the activation of antiMRP3 (scFv) -sTRAIL to two kinds of death receptors in U251 cells, the mRNA expression level of TRAIL-R1 and TRAIL-R2 in U251 cells was analyzed by real-time PCR method. U251 cell 1H was incubated with anti -TRAIL-R1 and anti -TRAIL-R2 antibodies, and TRAIL-R1 and TRAIL-R2 were blocked separately or simultaneously. Then U251 cell 24h was treated with IC50 dose antiMRP3 (scFv) -sTRAIL, and the apoptotic rate was detected by flow cytometry. The activation of two death receptors was analyzed. The apoptosis rate of L-R1 decreased by 28%, the apoptosis rate of blocking TRAIL-R2 decreased by 35%, and the apoptosis rate of TRAIL-R1 and TRAIL-R2 decreased 46%. at the same time. The Western blot method was used to verify that antiMRP3 (scFv) -sTRAIL fusion protein activates exogenous apoptosis pathway and induces apoptosis of caspase dependent cells.
The above results show that the specific binding of antiMRP3 (scFv) -sTRAIL fusion protein and target antigen transforms sTRAIL from soluble form into a membrane binding form of natural TRAIL. It can not only activate TRAIL-R1 but also restore its activation ability to TRAIL-R2, enhance the apoptosis inducing activity of sTRAIL, and thus obtain enhanced anti-tumor effect.AntiMRP3. (scFv) -sTRAIL fusion protein activates the exogenous apoptotic pathway by inducing the death of caspase dependent cells by binding to two death receptors, TRAIL-R1 and / or TRAIL-R2.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392;R739.41

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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7 張

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